Process for producing D-amino acids with composite immobilized enzyme preparation

ABSTRACT

A process for the efficient production of a D-amino acid from the corresponding DL-5-substituted hydantoin by one-step reaction which comprises using a composite immobilized enzyme at a pH about neutrality, said composite immobilized enzyme being obtained by immobilizing a hydantoinase having its optimal pH within an alkaline range and a D-N-carbamyl-α-amino acid amidohydrolase having its optimal pH about neutrality in a coexisting state on an immobilizing support, simultaneously, is disclosed.

This is a continuation of U.S. national stage application ofPCT/JP95/01257 filed Jun. 23, 1995.

FIELD OF THE INVENTION

The present invention relates to a composite immobilized enzymepreparation and a process for producing D-amino acids using thepreparation. In particular, the composite immobilized enzyme preparationof the present invention is useful for the production of D-α-amino acidswhich are intermediate compounds for the production of antibiotics, suchas D-(p-hydroxyphenyl)glycine to be used for the production of theantibiotic, amoxycillin and the like.

BACKGROUND OF THE INVENTION

Optically-active D-amino acids are important compounds as intermediatecompounds for drugs and it has been known that they can be producedefficiently by combining an asymmetric hydrolysis reaction of5-substituted hydantoins into the corresponding D-N-carbamyl-α-aminoacids with the enzymes, hydantoinases (hereinafter sometimes abbreviatedas "Hase") (JP-B 62-30785), and an conversion reaction of the resultantD-N-carbamyl-a-amino acids into the corresponding D-α-amino acids withthe enzymes, D-N-carbamyl-α-amino acid α-midohydrolases (hereinaftersometimes abbreviated as "decarbamylase" or "DCase") (PCT/JP91/01696: WO92/10579).

In addition, JP-A 63-185382, WO 92/00739 and the like disclose that therespective reactions are carried out more efficiently by using theseenzymes in the form of so- called immobilized enzymes wherein they areimmobilized on supports such as ion exchange resins and the like.

However, a two-step reaction has been employed for carrying out thesereactions because the optimal and stable pH's of both enzymes areconsiderably different from each other. Therefore, both immobilizedenzymes should be prepared separately and complicated reactionoperations are required.

OBJECTS OF THE INVENTION

The present invention relates to a technique for producing D-α-aminoacids efficiently by using an immobilized enzyme resin obtained byimmobilizing both hydantoinase and decarbamylase on one immobilizingresin support in a coexisting state of the enzymes (hereinafter referredto as a "composite enzyme"), simultaneously.

In these two enzymatic reactions for converting 5-substituted hydantoinsinto D-α-amino acids, in general, the optimal pH of the hydantoinasereaction is pH 8 to 9 and the solubility of the substrate is increasedas increase in pH. In addition, the racemic reaction of the hydantoinring is promoted in an alkaline range. Therefore, it is desired to carryout the hydantoinase reaction in the pH ranging from 7 to 10, preferablyin an alkaline range. On the other hand, in general, the optimal pH ofthe decarbamylase reaction is pH 6.5 to 9.0 but the hindrance of thereaction by ammonia formed is remarkably increased as increase in pH.Therefore, it is desired to carry out the decarbamylase reaction at pHabout neutrality.

If so-called one-step reaction can be employed for carrying out thesereactions, i.e., these two enzymatic reactions can be carried out in onereaction vessel simultaneously, in comparison with so-called two-stepreaction wherein two different enzymes react with the substratesseparately, reaction operations become simple and the overall reactiontime can be shortened. In addition, by combining the hydantoinasereaction which is a reversible reaction and the decarbamylase reactionwhich is a irreversible reaction, the conversion yield of hydantoin canbe improved as well as the subsequent purification of D-amino acids canbe simplified. Thus, it is expected to significantly reduce theproduction cost. However, when the respective enzymes are immobilized ondifferent immobilizing supports and they are mixed upon using them, thereactivities become inferior because of the difference in optimal pH andthere is a problem in the stability of the immobilized enzymes.

The present inventors have intensively studied to produce a compositeimmobilized enzyme preparation wherein both enzymes are immobilized onone immobilizing support simultaneously. If these two enzymes areimmobilized simultaneously, two enzymatic reactions occur successivelyin micro-spaces of resins. Therefore, movement of the substrate for adecarbamylase, a D-N-carbamyl-α-amino acid, from immobilized Hase toimmobilized DCase by diffusion is not required and pH variation in themicro-spaces can be minimized. Thus, it is expected that, in addition toincrease in reactivities of the respective enzymes and relief of theproduct hindrance due to ammonia, stability of enzymes upon usingrepeatedly is improved.

SUMMARY OF THE INVENTION

The present invention provides a process for the efficient production ofa D-amino acid from the corresponding DL-5-substituted hydantoin byone-step reaction which comprises using a composite immobilized enzymeat a pH about neutrality, said composite immobilized enzyme beingobtained by immobilizing a hydantoinase having its optimal pH within analkaline range and a D-N-carbamyl-α-amino acid amidohydrolase having itsoptimal pH about neutrality in a coexisting state on an immobilizingsupport of an anionic ion exchange resin, simultaneously (hereinafterreferred to as composite enzyme).

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a graph illustrating the time courses of the reactions for theproduction of the corresponding D-p-hydroxyphenylglycine from5-(p-hydroxyphenyl)hydantoin in Example 3 by using the compositeimmobilized enzyme preparation of the present invention and the mixtureprepared by immobilizing hydantoinase and decarbamylase separately tothe different resins.

FIG. 2 is a graph illustrating the stability of the enzymes in repeatedreactions by using the composite immobilized enzyme preparation inExample 4.

FIG. 3 is a graph illustrating the comparison of the activity of thecomposite immobilized enzyme preparation with the activity of themixture of the separately prepared immobilized enzyme preparations (thehydantoinase activity is twice as much as the composite immobilizedenzyme preparation) in Example 6.

DETAILED EXPLANATION OF THE INVENTION

As for the hydantoinase used in the present invention, the enzymes fromanimals, plants and microorganisms can be used. The hydantoinases frommicroorganisms are suitable for industrial production. As such amicroorganism, those disclosed in JP-B 62-30758 are exemplified. Thebacteria include Achromobacter, Aerobacter, Aeromonas, Aqrobacterium,Alcaliqenes, Arthrobacter, Bacillus, Brevibacterium, Corynebacterium,Enterobacter, Erwinia, Escherichia, Klebsiella, Microbacterium,Micrococcus, Protaminobacter, Proteus, Pseudomonas, Sarcina, Serratia,Xanthomonas and the like. The Actinomycetes include Actinomyces,Mycobacterium, Nocardia, Streptomyces, Actinoplanes and the like. Thefilamentous fungi include Asperqillus, Paecilomyces, and Penicillium andthe like. The yeasts include Candida, Pichia, Rhodotorula, Torulopsisand the like.

Among the above microorganisms, examples of strains which haverelatively high hydantoinase activities and are excellent in practicaluse include Aerobacter cloacae IAM 1221, Agrobacterium rhizogenes IFO13259, Brevibacterium incertum IFO 12145, Corynebacterium sepedonicumIFO 3306, Microbacterium flavum ATCC 10340, Micrococcus roseus IFO 3764,Pseudomonas striata IFO 12996, Mycobacterium smecmatis ATCC 607,Nocardia corallina IFO 3338, Streptomyces flaveolus IFO 3241, Bacillussp. KNK108 (FERM P-6056), Bacillus sp. KNK245 (FERM BP-4863) and thelike.

In addition, the enzymes produced by artificial microorganisms in whichhydantoinase productivity is imparted or increased by gene recombinanttechnique, for example, E. coli HB101pTH104 (FERM BP-4864), or thedihydropyrimidinase having similar activity as disclosed in JP-A53-136583 can be used.

As for the decarbamylase used in the present invention, the originsthereof is not specifically limited and those derived from animals,plants and microorganism can be used. However, for industrialproduction, the enzymes from microorganisms are suitable. Examples ofsuch microorganisms include naturally occurring microorganisms such asAgrobacterium Pseudomonas, Arthrobacter, Alcaligenes, Achromobacter,Moraxella, Paracoccus, Aerobacter, Aeromonas, Brevibacterium, Bacillus,Flavobacterium and Serratia disclosed in JP-B 57-18793, JP-B 63-20520and JP-B 1-48758, or the artificial microorganisms described in WO91/01696 in which decarbamylase productivity is imparted or increased bygene recombinant technique. Representative examples of suchmicroorganisms include Agrobacterium sp. KNK712 (FERM BP-1900),Pseudomonas sp. KNK003A (FERM BP-3181), Pseudomonas sp. KNK505 (FERMBP-3182), Escherichia coli JM109pAD108 (FERM BP-3184), E. coliJM109pPD304 (FERM BP-3183) and the like.

When a stabilized decarbamylase in which the amino acid responsible forheat resistance of the decarbamylase is replaced by another one is used,the following transformants disclosed in WO 94/03613 can be used. Forexample, the transformants include E. coli JM109pAD402 (FERM BP-3912),E. coli JM109pAD404 (FERM BP-3913), E. coli JM109pAD406 (FERM BP-3914),E. coli JM109pAD416 (FERM BP-3915), E. coli JM109pAD428, E. coliJM109pAD429 (FERM BP-4035), E. coli JM109pAD431, E. coli JM109pAD434, E.coli JM109pAD435, E. coli JM109pAD439, E. coli JM109pAD441, E. coliJM109pAD445, E. coli JM109pAD447, E. coli JM109pAD448, E. coliJM109pAD450, E. coli JM109pAD421, E. coli JM109pAD422, E. coliJM109pAD423, E. coli JM109pAD424 (FERM BP-4034), E. coli JM109pAD425, E.coli JM109pAD426, E. coli JM109pAD427, E. coli JM109pAD451, E. coliJM109pAD452, E. coli JM109pAD453, E. coli JM109pAD461, E. coliJM109pAD454, E. coli JM109pAD455 (FERM BP-4036), E. coli JM109pAD456, E.coli JM109pAD468, E. coli JM109pAD469, E. coli JM109pAD470, E. coliHB101pNT4553 (FERM BP-4368) or the like. When such stabilized enzymesare used, better results can be obtained in repeated use of thecomposite immobilized enzyme preparation of the present invention.

The enzymes used in the present invention can be produced simultaneouslyby using a microorganism which is capable of producing both enzymes.Alternatively, the enzymes can be produced separately or simultaneouslyusing microorganisms which are capable of producing respective enzymesalone. As the microorganism which is capable of producing both enzymes,a microorganism isolated from a natural source such as an Aqrobacteriumdisclosed in JP-B 63-250520 and the like can be used. In addition, arecombinant microorganism prepared by isolating genes of both enzymesfrom the microorganisms isolated from a natural source and introducingthem into a host, for example, the microorganisms disclosed in WO94/00577, can be used. Alternatively, genes of both enzymes can beisolated from the same or different microorganisms and inserted into thesame vector in the expressible form of both genes, or inserted intodifferent vectors having different replication modes, for example, pUC19and pACYC184. Then, the recipient host such as E. coli can betransformed with the above vector or vectors so that a singlemicroorganism can produce both enzymes. In these cases, by selecting thekinds of promoters having various capabilities and the plasmids havingdifferent copy numbers, the ratio of production of each enzyme can bevaried according to a particular purpose. However, it is preferred toadjust the ratio so as to obtain almost equal amounts of enzymeproteins. In the case that microorganisms which produce differentenzymes separately are used, strains isolated from natural sources orrecombinant microorganisms prepared by using gene recombinant techniquecan also be used. In such case, the production of the enzymes can becarried out either by cultivating the producer microorganismsseparately, or cultivating in a mixed culture at various ratios,preferably, at such a ratio that almost equal amounts of the enzymes canbe produced.

The cultivation can be carried out aerobically, for example, by shakingculture using flasks or by spinner culture with aeration. As for culturemedium used in the cultivation, normally, an nutrient medium whichcontains generally used natural nutrients such as meat extract andpolypeptone and the like can be used. When hydantoinase is producedseparately or simultaneously with decarbamylase, a good result can beobtained by carrying out the cultivation with addition of manganese ionin an amount of, for example, as manganese chloride, 1 to 100 mg/liter,preferably about 20 mg/liter.

In the present invention, the enzymes can exist in the immobilizedenzyme preparation in the purified, partially purified or crude form, orin some cases, in the form of microbial cells per se. And, in so far asthe enzymes are under the conditions that the enzymatic activities canbe exhibited the enzymes can exist in any form, and can be accompaniedby any substance.

In the preparation or use of the composite immobilized enzymepreparation of the present invention, better results can be obtained byadding an antioxidant for a repeated use. As such an antioxidant, therecan be used dithiothreitol, 2-mercaptoethanol, L-cystein hydrochloride,cysteamine hydrochloride, dithioerythritol, a mixture of dithiothreitoland dithioerythritol, reduced glutathione and the like.

An immobilized support used can be varied according to particular useconditions of the immobilized enzymes. When a crude enzyme solution suchas a cell-free extract, or a partially purified enzyme solution treatedby ammonium sulfate precipitation or the like are subjected toimmobilization, polymer supports having ion exchanging groups orcovalent bonding groups can be used.

As for a polymer support having an ion exchanging group, for example,Duolite A (registered trade mark) series, or Amberlite IRA (registeredtrade mark) series, the exchanging groups of which are primary,secondary, tertiary and quaternary amines; or a polystyrene resin havinga diethanol type functional group, for example, Diaion EX can be used.

As for a support with a covalent bonding group, a substitutedpolymethacrylate polymer having an aldehyde as a bonding group, a highdensity alumina covered with a complex ofpolyethyleneimine/glutaraldehyde and the like can be used.

To immobilize whole microbial cells such as live cells or dried cells, apolymer such as polyacrylamide, polyurethane or calcium alginate, or aporous material such as alumina can be used.

The composite immobilized enzyme preparation of the present invention isproduced as follows. A solution of the crude composite enzyme in whichactivities of Hase and DCase are appropriately adjusted is contactedwith a support to adsorb the respective enzymes and treated with across-linking agent for stabilization. To prepare a solution of thecrude composite enzyme, firstly, each enzyme is produced by cultivatingmicrobial cells. In this case, a cell-free extract can be prepared bycollecting the cells and disrupting them with, for example, sonication,mechanical disruption (for example, homogenizer) or enzyme treatment,when a microorganism capable of producing the enzymes simultaneously isused. When a solution of the composite enzyme is prepared by usingdifferent microorganisms, it can be prepared by cultivating themicroorganisms separately, collecting the cells, preparing respectivecell-free extracts and mixing them. Or, it can be prepared bycultivating respective producer microorganisms together and disruptingthe cells at the same time to prepare a cell-free extract in which bothtwo enzymes are mixed. When both enzymes are produced by differentmicroorganisms, the productivities vary to a large extent according tothe kinds of the microorganisms and difference in the cultivationmethods. However, in general, regarding hydantoinase, a normal producermicroorganism produces hydantoinase at about 0.1 to about 10 units/ml(one unit of the enzyme used herein is defined as an amount required toconvert the substrate 5-(p-hydroxyphenyl)hydantoin intoD-N-carbamyl-p-hydroxy-phenylglycine at pH 8.7, 40° C., for 1 min) and arecombinant artificial producer microorganism produces it at about 5 toabout 150 units/ml. Regarding decarbamylase, a normal producermicroorganism produces decarbamylase at about 0.01 to about 2 units/ml(one unit of the enzyme used herein is defined as an amount required toconvert the substrate D-N-carbamyl-p-hydroxyphenylglycine intoD-p-hydroxyphenylglycine at pH 7.0, 40° C., for 1 min) and a recombinantartificial producer microorganism produces it at about 0.1 to about 20units/ml. The ratios of Hase and DCase activities in a solution of thecrude composite enzyme are adjusted so that the reaction producingD-α-amino acid from 5-substituted hydantoin can proceed mostefficiently. As described hereinafter, this reaction is carried out at apH of neutrality which is near the optimal pH for DCase and thereforethe conditions are different from those for exhibiting maximal activityof Hase. Then, it is desired that both enzymatic activities are adjustedso that almost equal activities are exhibited at the reaction pH. Forexample, in the case of carrying out the reaction at pH 7.5, the desiredcomposite immobilized enzyme preparation can be produced by carrying outthe immobilization reaction using an enzyme solution in which there arealmost equal amounts of proteins and enzymatic activities so thathydantoinase is about five times as much as the DCase in units. Tomaintain such activities after immobilization, it is desired that theratio of the enzyme activities (Hase: DCase) in a solution of the crudecomposite enzyme should be in a range of 1 to 10:1. Therefore, afterdisruption of the microbial cells and before adsorption, the activitiesof the crude composite enzyme are adjusted to such levels.

In addition, to adsorb appropriate amounts of enzymes on the support, itis preferred to adjust the concentration of decarbamylase in thesolution of the crude composite enzyme to 10 to 300 units/ml. Theactivity adsorbed is about 20 to about 90% of the activity added and nosubstantial difference can be seen between both enzymes.

The support is used after activation of its exchanging group with, forexample, aqueous solution of sodium chloride and equilibrating, forexample, in a buffer solution. It is preferred that the ratio of thesolution of the crude composite enzyme and the support is adjusted sothat the total amount of the protein in the crude enzyme solution arealmost the same as the maximum adsorption capacity of the support. Ifnecessary, 1 to 10 mM of an antioxidant and/or 0.5 to 20 mM of manganeseion can be present. The mixture is stirred at 4 to 30° C., preferably at15° C., and the support is collected by filtration after the amount ofenzyme adsorbed reaches the given amount (normally 8 to 48 hours,preferably this is carried out under the atmosphere of inert gas, suchas nitrogen). Then, the support is washed and insolubilized by treatingwith a cross-linking agent to stabilize it. As for a cross-linkingagent, a known agent such as at less than 1%, preferably 0.1 to 0.2%glutaraldehyde can be used. The cross-linked composite enzymeimmobilized preparation is washed with distilled water and a buffersolution (preferably containing 0.1 to 20 mM, normally 1 to 5 mMantioxidant as described above) and stored in wet state in a sealedvessel at a low temperature (4° C.). In general, the activities of thecomposite immobilized enzyme preparation thus obtained are 5 to 80units/g support with respect to decarbamylase. For hydantoinase, itsactivity is 1 to 10 times as much as the decarbamylase activityaccording to the adsorption conditions.

The process for producing D-a-amino acids from 5-substituted hydantoinsby using the composite immobilized enzyme preparation of the presentinvention will be described hereinafter.

The reaction is carried out by reacting the substrate, 5-substitutedhydantoin, with the composite immobilized enzyme preparation, ifnecessary, in the presence of an antioxidant agent and/or manganese ion.Normally, it is preferred that the reaction is carried out in thepresence of 0.1 to 20 mM of an antioxidant agent and manganese ion. Thereaction proceeds according to the following reaction scheme: ##STR1##wherein R represents phenyl group, phenyl group substituted with hydroxygroup, alkyl group, substituted alkyl group, aralkyl group or thienylgroup.

The concentration of the substrate 5-substituted hydantoin to be used is0.1 to 30% (w/v), preferably 1 to 5% (w/v). It is preferred that theamount of the composite immobilized enzyme preparation to be used isabout 10 to about 20 units/g substrate as decarbamylase activity. Thereaction temperature varies according to particular enzymes but, ingeneral, it is 30 to 60° C. Enzymes having heat resistance be used atmuch higher temperature.

The reaction pH is suitably selected from the range of 6.5 to 8.0. ThepH range is almost optimal for DCase. However, it is considerably farfrom the optimal pH for Hase and the Hase activity decreases severaltimes as low as the activity at the optimal pH. The rate of racemizationalso decreases. However, ultimately, the production of D-α-amino acid isgoverned by the enzyme of the last step, DCase, it is preferred to setthe amount of the enzyme and the reaction conditions based on the DCase(i.e., the optimal conditions for DCase) and to combine Hase activity inproportion to that DCase activity. In general, the ratio of the enzymesto be adsorbed to the support is adjusted so that almost equalactivities can be expressed, although this varies according toparticular reaction conditions. The "almost equal activities" mean thesituation where the ratio of the hydantoinase activity measured at pH7.5 (represented in "units (pH 7.5)"), and the decarbamylase activity asdescribed above is about 0.5 to 1.5:1. The reaction is carried out withadjusting a reaction pH to the pH range thus selected, normally to pH7.5. By these operations, the unfavorable conditions for Hase areovercome and the reaction equilibrium is declined toward the productionof D-N-carbamyl amino acid. Thus, the reaction can be carried out moreefficiently than expected and the conversion of the substrate, a5-substituted hydantoin, can be improved. When the reaction is carriedout at an alkaline pH range near the optimal pH of hydantoinase, thedecarbamylase activity decreases to below the half of the activity atthe optimal pH, though the production of a D-N-carbamyl amino acid canproceed successfully. In addition, it has been observed that the wholeyield decreases and that the stability of decarbamylase itself islowered because the reaction is largely inhibited by ammonia produced bythe reaction. For these reasons, the reaction using the compositeimmobilized enzyme preparation can be carried out efficiently when thecomposite immobilized enzyme preparation having such immobilizationratio that the decarbamylase reaction is slightly rate-determining isused under conditions near the optimal reaction conditions and thesubstrate concentration of decarbamylase. In addition, it isadvantageous that the whole activities of the composite immobilizedenzyme preparation is easily controlled because the productivity of Haseis high and the DCase catalyzes the irreversible reaction.

The reaction is carried out normally by column method or by suspendingthe composite immobilized enzyme preparation in a reaction vessel. Inthe latter case, a batch reaction is usually carried out and thereaction time is about 6 to about 48 hours per batch. By using thecomposite immobilized enzyme preparation of the present invention, thecorresponding D-amino acid can be produced from a 5-substitutedhydantoin at a high yield in a one-step reaction. The following examplesfurther illustrate the present invention in detail but are not to beconstrued to limit the scope thereof.

EXAMPLE 1

Firstly, for preparing an enzyme solution of hydantoinase, Bacillus sp.KNK108 (FERM P-6056) was inoculated to a 250 ml of seed culture medium(meat extract 1.0%, polypeptone 1.0%, yeast extract 0.5% (pH 7.0)) andcultivated at 33° C. for about 25 hours. This seed culture wasinoculated to 2.5 liter of a culture medium (meat extract 1.0%,polypeptone 1.0%, yeast extract 0.5%, uracil 0.1%, MnCl₂ 20 ppm (pH7.5)) and cultivated at 33° C. for about 16 hours. The microbial cellswere collected by centrifugation and suspended in 50 ml of 20 mM MnSO₄aqueous solution. After adjusting the pH to 8.5, the cells weredisrupted by sonication and the residue was removed to obtain a crudeenzyme solution of hydantoinase (107 units/ml).

In addition, to prepare an enzyme solution of decarbamylase, E. coliHB101pNT4553 (FERM BP-4368) was cultivated in 20 ml of 2YT medium (Bactopeptone 1.6%, Bacto yeast extract 1.0%, NaCl 0.5%) supplemented with 50μg/ml ampicillin at 37° C. for about 16 hours. This culture wasinoculated to 1.4 liter of 2YT medium in an amount of 1%, and cultivatedat 37° C. for about 28 hours. The cells were collected by centrifugationand suspended to 140 ml of 5 mM dithiothreitol solution. After adjustingthe pH to 7.0, the cells were disrupted by sonication and the residuewas removed to obtain the supernatant as a crude enzyme solution ofdecarbamylase (36 units/ml).

EXAMPLE 2

By using the crude enzyme solutions obtained in Example 1,immobilization of the enzymes was carried out. Duolite A-568 (Rohm &Haas) as a immobilization support was washed with firstly 1M NaCl anddeionized water and then put into the deionized water and the pH wasadjusted to 7.5. To 8.4 g of this resin were added 20 ml of the crudeenzyme solution of hydantoinase and 11.5 ml of the crude enzyme solutionof decarbamylase the pH of both of which had been adjusted to 7.5 andthe mixture was stirred at 15° C. for about 20 hours under nitrogenatmosphere. After washing this resin twice with 0.5 mM MnSO₄ solution,the resin was suspended in five time volumes of deionized water. Afterthe pH was adjusted to 7.5, the suspension was stirred for 35 minuteswith addition of 544 μl of 2.5% glutaraldehyde with portions. Thissuspension was treated with 50 mM Tris-HCl (pH 7.5), 5 mM DTT and 1 mMMnSO₄ overnight and then the composite immobilized enzyme preparationwas collected by filtration (hydantoinase activity 8.2 units (pH 7.5)and decarbamylase activity 9.9 units per 1 g resin).

Then, as controls, resins to which two enzymes were separatelyimmobilized were prepared.

Each of 20 ml of the crude enzyme solution of hydantoinase and 60 ml ofthe crude enzyme solution of decarbamylase was mixed with each of 4.2 gand 22.0 g of the above immobilization support and the same operationsas described above was carried out to obtain each immobilized enzymeresin in which each enzyme was separately immobilized (hydantoinaseimmobilized enzyme: 24 units/g resin, 2.7 units/g resin (pH7.5), anddecarbamylase immobilized enzyme: 37 units/g resin).

EXAMPLE 3

By using the composite immobilized enzyme preparation and controlimmobilized enzymes containing respective single enzymes obtained inExample 2, the reactions to produce the corresponding D-α-amino acidfrom a 5-substituted hydantoin were carried out.

After addition of 1g of 5-(p-hydroxyphenyl)hydantoin as the substrate to100 ml of 0.1 M KPB (pH 7.5), 1 MM MnSO₄ and 5 mM DTT, nitrogen gas wassufficiently bubbled in and the reaction conditions were adjusted to 40°C. and pH 7.5. In order to obtain the same enzymatic activities of bothenzymes, 0.97 g of the composite immobilized enzyme preparation or 3.0 gof the hydantoinase immobilized enzyme and 0.26 g of the dacarbamylaseimmobilized enzyme were added. The reaction was carried out withbubbling of nitrogen gas and controlling the reaction pH to 7.5 with 2NH₂ SO₄ or 6N NaOH. The reaction was carried out for 29 hours withperiodical samplings. The amount of p-hydroxyphenylglycine produced ateach sampling point was determined by high performance liquidchromatography (Nippon Bunko, Finepack SIL C-18 column).

The results are shown in FIG. 1.

As is seen from FIG. 1, it is found that the reaction can be carried outmore efficiently when two enzymes were immobilized simultaneously on thesame resin.

EXAMPLE 4

The stability of the enzyme activities was investigated by carrying outthe reaction repeatedly with the composite immobilized enzymepreparation. Two grams of 5-(p-hydroxyphenyl)hydantoin was added to 100ml of 1 mM MnSO₄ and 5 mM DTT and pH was adjusted to 7.5. To the mixturewas added 8.9 g of the composite immobilized enzyme preparation obtainedin Example 2 was added and the reaction was carried out at 40° C. for 23hours with bubbling of nitrogen gas and controlling the pH to 7.5. Afterfiltering the reaction mixture with suction, another mixture was addedto the composite immobilized enzyme according to the same manner asdescribed above and the reaction was carried out. This operation wasrepeated five times and both enzyme activities were determined at theend of each reaction.

The relative activities to those in the first reaction are shown in FIG.2.

The decrease in the activities were scarcely observed in these fivereactions.

EXAMPLE 5

For preparing an enzyme solution of hydantoinase, E. coli HB101pTH104(FERM BP-4864) containing a hydantoin gene from Bacillus sp. KNK245(FERM BP-4863) was cultivated in 20 ml of 2YT medium at 37° C. for about16 hours. This culture was transferred to 1.2 liter of 2YT mediumsupplemented with 50 μg/ml of ampicillin and 400 ppm of MnCl₂.4H₂ O andcultivated for 26 hours at 37° C. The cells were collected bycentrifugation and suspended in 80 ml of 1 mM MnSO₄ aqueous solution.After adjusting the pH to 8.5 with ammonia water, the cells weredisrupted by sonication and the residue was removed by centrifugation.After adjusting the pH of the supernatant to 8.5, heat treatment at 60°C. was carried out for 20 min. The denatured proteins were removed bycentrifugation to obtain a crude enzyme solution of hydantoinase (1,100units/ml).

By using this crude enzyme solution and a crude enzyme solution ofdecarbamylase prepared according to the same manner as described inExample 1 (240 units/ml), the composite immobilized enzyme preparationwas prepared according to the same manner as described in Example 2. Byusing 30 ml of the crude enzyme solution of decarbamylase and 13 ml ofthe crude enzyme solution of hydantoinase, the enzymes were immobilizedon 29 g of the resin according to the same manner as described inExample 2 (decarbamylase: 45 units, and hydantoinase: 119 units, 44units (pH 7.5) per 1 g of resin)

As controls, resins on which two enzymes were separately immobilizedwere used. The decarbamylase immobilized enzyme (43 units/g-resin) wasprepared as described in Example 2. The hydantoinase immobilized enzymewas prepared according to the method described in Example 2 by mixing21.8 g of the resin for immobilization with 60 ml of the crude enzymesolution (177 units/g-resin, 51 units/g (pH 7.5))

EXAMPLE 6

By using 5 g of the composite immobilized enzyme preparation obtained inExample 5, and as immobilized enzymes prepared by immobilizingrespective enzymes on different resins, a mixture of 5.6 g of thehydantoinase immobilized enzyme and 5.2 g of the decarbamylaseimmobilized enzyme obtained in Example 5 (the decarbamylase activity (pH7.5) was equal to the composite immobilized enzyme preparation and thehydantoinase activity (pH 8.7) was twice as much as the compositeimmobilized enzyme preparation), the reaction was carried out with 3%substrate according to the method described in Example 3.

The results are shown in FIG. 3.

As shown in FIG. 3, the reactivity of the composite immobilized enzymepreparation was similar to the reactivity of the separately immobilizedenzymes in spite that the hydantoinase activity was one half of theseparately immobilized enzyme. Thus, it has been found that the reactionby the composite immobilized enzyme preparation is more efficient andthat the amount of the expensive resin for immobilization can be largelyreduced.

EXAMPLE 7

By using each 5 g of the composite immobilized enzyme obtained inExample 5, the effect of the reaction pH was investigated. In three pHlevels of 7.0, 7.25 and 7.5, the reactions with 3% substrate werecarried out. The times required to convert 99% of the substrate wasmeasured. The times were 5.5, 5.4 and 6.75 hours, respectively. Thus, ithave been found that pH 7.0 and 7.25 are advantageous to the reaction.

As described hereinabove, according to the present invention, by usingof the composite immobilized enzyme preparation produced by immobilizingthe hydantoinase and the decarbamylase simultaneously on the same resinin the production of the corresponding D-α-amino acids from5-substituted hydantoins, it is possible to carry out the one-stepreaction with much simpler operations than two-step reaction and moreefficiently than the reaction by the mixture of the two immobilizedenzymes obtained by immobilizing these enzymes separately to thedifferent resins.

What is claimed is:
 1. A process for the production of a D-aminio acidfrom the corresponding DL-5-substituted hydantoin which comprisesreacting the 5-substituted hydantoin in a concentration of
 0. 1 to 30%(w/v) with a composite immobilized enzyme preparation having about 10 toabout 20 units/g 5-substituted hydantoin as DCase activity at a pH ofabout from 6.5 to 8.0, wherein said composite immobilized enzymepreparation comprises an immobilized hydantoinase (Hase) having itsoptimal pH within an alkaline range and an immobilizedD-N-carbamyl-α-amino acid amidohydrolase (DCase) having its optimal pHabout neutrality, wherein the ratio of said Hase activity to said DCaseactivity of said composite immobilized enzyme is about 0.5-1.5 to 1.0 asunit of activity measured at pH 7.5, and wherein said compositeimmobilized enzyme preparation is prepared by adsorbing said amount ofHase and DCase on and immobilized support in a solution of crudecomposite enzymes, said solution containing Hase and DCase at a ratio ofHase to DCase of about 1-10 to 1 and containig DCase in a concentrationof about 10 to 300 units/ml as a unit of activity measured at pH 7.5 andwhereby the said Has a said DCase are immobilized on a simple resin. 2.A process for the production of a D-amino acid according to claim 1,wherein said Hase is that derived from a microorganism belonging to thegenus Bacillus.
 3. A process for the production of a D-amino acidaccording to claim 1, wherein said Hase is that derived from Bacillussp. KNK108 (FERM BP-887), Bacillus sp. KNK245 (FERM BP-4836) orAerobacter cloacae IAM 1221 (FERM BP-1898).
 4. A process for theproduction of a D-amino acid according to claim 1, wherein said Hase isderived from E. coli HB101pTH104 (FERM BP-4864).
 5. A process for theproduction of a D-amino acid according to claim 1, wherein said DCase isderived from a microorganism selected from the group consisting ofAgrobacterium sp. KNK712 (FERM BP-1900), Pseudomonas sp. KNK003A (FERMBP-3181), Pseudomonas sp. KNK505 (FERM B-P-3182), E. coli JM109pAD108(FERM BP-3184), and E. coli JM109pPD304 (FERM BP-3183).
 6. A process forthe production of a D-amino acid according to claim 1, wherein saidDCase is that derived from a recombinant microorganism selected from thegroup consisting of E. coli JM109pAD402 (FERM BP-3912), E. coliJM109pAD404 (FERM BP-3913), E. coli JM109pAD406 (FERM BP-3914), E. coliJM109pAD416 (FERM BP-3915), E. coli JM109pAD429 (FERM BP-4035), E. coliJM109pAD421, E. coli JM109pAD422, E. coli JM109pAD423, E. coliJM109pAD424 (FERM BP-4034, E. coli JM109pAD426, E. coli JM109pAD427, E.coli JM109pAD451, E. coli JM109pAD455 (FERM BP-4036), or E. coliJM109pAD4553 (FERM BP-4368), the stability of which is improved by aminoacid substitution of the DCase derived from Agrobacterium sp. KNK712(FERM BP-1900).
 7. A process for the production of a D-amino acidaccording to claim 1, wherein said composite enzyme is produced bycultivating said Hase producer microorganism and said DCase producermicroorganism together.
 8. A process for the production of D-amino acidaccording to claim 1, wherein said composite enzyme used forimmobilization is prepared by producing said Hase and said DCasesimultaneously by cultivating a microorganism selected from the groupconsisting of Agrobacterium sp. KNK-712 (FERM BP-1900), Rhizobium sp.KNK1415 (FERM BP=4419), Pseudomonas sp. KNK003A (FERM BP-3181), E. coliHB101pPHD301 (FERM BP-4866) and E. coli JM109pAHD101.
 9. A process forproduction of a D-amino acid according to claim 1, wherein said Hase isthat derived from a microorganism selected from the group consisting ofAgrobacterium radiobacter IFO 13259, Brevibactenun incertum IFO 12145,Microbacternum flavum ATCC 10340, Micrococcus roseus IFO 3764,Pseudomonas striata IFO 12996 and Mycobacterium smegmatis ATCC
 607. 10.A composite immobilized enzyme preparation comprising an Hase and aDCase, wherein said Hase and DCase are derived from the same ordifferent microorganisms; an antioxidant; manganese ion and a supportcarrying said enzymes, land wherein the ratio of said Hase activity tosaid DCase activity in said composite is about 0.5-1.5 to 1.0 as a unitof activity measured at pH 7.5 and said DCase activity is about 5 to 80units/g support, wherein said composite immobilized enzyme preparationis prepared by adsorbing said amount of Hase and DCase on an immobilizedsupport in a solution of crude composite enzymes, said solutioncontaining Hase and DCase at a ratio of Hase to DCase of about 1-10 to 1and containing DCase in a concentration of about 10 to 300 units/ml as aunit of activity measured at pH 7.5 and wherein said Has and said DCaseare immobilized on a single resin.
 11. A process for the production of aD-amino acid from the corresponding DL-5-substituted hydantoin whichcomprises reacting the 5-substituted hydantuin in a concentration of 0.1to 30% (w/v) with a composite immobilized enzyme preparation havingabout 10 to about 20 units/g 5-substituted hydantoin as DCase activityat a pH of about from 6.5 to 8.0, wherein said composite immobilizedenzyme preparation comprises an immobilized hydantoinase (Hasc) havingits optimal pH within an alkaline range and an immobilizedD-N-carbamyl-α-amino acid amidohydrolase (DCase) having its optimal pHabout neutrality, wherein the ratio of said Hase activity to said DCaseactivity of said composite immobilized enzyme is about 0.5-1.5 to 1.0 asunit of activity measured at pH 7.5, and wherein said Hase and saidDCase are immobilized on said support so that both enzymatic reactionsoccur successively in microspaces of resins.
 12. A composite immobilizedenzyme preparation comprising an Hase and a DCase, wherein said Hase andDCase are derived from the same or different microorganisms; anantioxidant; manganese ion and a support carrying said enzymes, whereinthe ratio of said Hase activity to said DCase activity in said compositeis about 0.5-1.5 to 1.0 as a unit of activity measured at pH 7.5 andsaid DCase activity is about 5 to 80 units/g support, and wherein saidHase and said DCase are immobilized on said support so that bothenzymatic are actions occur successively in micro-spaces of resins.